This proposal is in response to a request for applications to address "Genomic Profiling of Mental Disorders," and proposes intensive genomic profiling and functional analysis of Contactin Associated Protein 2 (CNTNAP2) as well as the presynaptic cytomatrix protein Piccolo (PCLO), in an effort to clarify their roles in Autism Spectrum Disorders (ASD). In addition, the application proposes to deeply sequence genes in the Contactin and Contactin Associated pathways, as well as genes coding for proteins known to interact with PCLO. We propose to leverage ongoing collaborations among the State, De Camilli and Giraldez labs at Yale to allow for rapid functional assays (both in vitro and in vivo) of identified rare sequence mutations, which will be used to clarify genomic findings and to confirm the relevance of rare mutations for disease risk. The organizing principles supporting this application are as follows: 1) Rare genetic variation is likely to play a significant role in the etiology of ASD;2) Methods of detecting rare (MAF<5%) and very rare (MAF<1%) variation in large numbers of patients and controls are now economically and technically feasible;3) Rare and very rare variations, including those in highly conserved coding regions, are widespread throughout the genomes of both affected and unaffected individuals;4) Rare and very rare alleles of intermediate effect will likely require association as opposed to linkage strategies to evaluate disease risk;and 5) That among the most pressing issues at this stage in the success of genomic profiling of mental disorders are differentiating disease-related mutations from low frequency neutral mutations and addressing the methodolgical challenges of assessing disease association in the case of rare and very rare alleles. This application proposes to address these issues as follows: 1) Deep-resequencing using next generation technologies to identify rare and very rare mutations in a large, extremely well-characterized and carefully ethnically matched case-control sample;2) To use quantitative ASD phenotyping in addition to categorical diagnoses in an effort to enhance the power of case-control association of rare variants;3) To leverage well established association methodologies, including genomic control, mutation burden analyses for very rare mutations, correction for multiple comparisons and replication samples, to decrease type 1 error;4) To use both in vitro and in vivo methods to evaluate the functional consequences of rare mutations identified in cases and controls, specifically in the genes CNTNAP2 and PCLO;and importantly, 5) To test the hypothesis that functional assays stratifying rare functional from neutral variation in CNTNAP2 will clarify the results of association analyses, as has been successfully employed in other rare variant studies (1). PUBLIC HEALTH RELEVANCE: This study seeks to identify the role of specific genes in Autism Spectrum Disorders. The research plan utilizes high throughput sequencing technologies and combines these with molecular in vitro and in vivo biological studies in an effort to clarify the precise contribution of two brain expressed molecules Contactin Associated Protein 2 and Picollo as well as related genes to Autism and related conditions.